The present invention relates to a transfecting peptide vector, to a composition containing the said vector as well as to their applications in the treatment (medicaments) and the prevention (vaccines) of human and animal diseases. The said vector is in particular capable of dispensing to suitable target cells nucleic sequences, proteins, peptides and chemical substances of interest.
In the field of gene therapy, many compositions useful for efficiently transfecting eukaryotic cells with a selected genetic material have been described.
There are essentially two main types of transfection vectors:
the natural transfection vectors, such as viruses or modified viruses, which are efficient but which have limits to their use: tissue nonspecificity, necessity to obtain constructs for each gene of interest and potential risks for the environment which lead to the setting in place of costly and constraining clinical infrastructures for the patient and staff;
nonviral agents (synthetic vectors), capable of promoting the transfer and the expression of chemical substances such as DNA into eukaryotic cells. The latter strategy represents an alternative to viral vectors.
These synthetic vectors must essentially have two functions: to condense the DNA to be transfected and to promote its cellular attachment as well as its passage across the plasma membrane and possibly the nuclear membranes; such vectors must therefore mimic the functioning of viruses in order to be efficient; however, it appears that the different vectors provided in the prior art do not exhibit these two functions in an optimum manner and may, in addition, depending on the cases, be toxic for the cells.
Among these nonviral agents, there may be mentioned first of all the cationic polymers and the cationic lipids. The former generally consist of polylysine, whereas a wide variety of cationic lipids (liposomes or pseudoliposomes) exist, each giving transfection efficiencies which vary according to the cell types (DOTMA, and the like).
The lipid portion which interacts with and/or destabilizes the membranes allows the fusion and the entry of the DNA/liposome complex.
However, the transfection of DNA by liposomes, although less immunogenic than that performed with the aid of cationic polymers, is in fact a relatively inefficient method.
The major mechanism of entry of the DNA/liposome complexes is, it seems, endocytosis; consequently, the transfected DNA is trapped in the intracellular vesicles and destroyed by the lysosomal enzymes.
Even if a portion of the transfected DNA is released into the cytoplasm by a mass action effect, only a small fraction of this DNA is effectively present in the nucleus.
Agents capable of increasing the release of the DNA from the endosomal vesicles and its passage into the nucleus can increase the gene transfer rate.
Among these agents, there may be distinguished:
those which target the complex towards another point of entry: the targeting is obtained, for example, by coupling ligands with the polylysine polymers; the targeting can also occur after internalization, by directing the complexes to the nucleus (PCT International Application WO 95/31557), and
those which avoid endosomal degradation; to escape endosomal degradation, it has been proposed to incorporate an endosomolytic agent into the complex, such as adenoviral particles (PCT International Application WO 93/07283) or more recently synthetic peptides with endosomolytic activity, which increase the release of the DNA into the cytoplasm.
Taking into account the preceding text, various types of complexes have been provided; there may be mentioned complexes combining liposomes and peptides, such as those described in:
International Application WO 96/25508, which describes compositions comprising (i) the nucleic acid to be transfected, (ii) a transfection agent, such as a cationic polymer and/or a lipofectant, (iii) a peptide compound involved at the level of the condensation of the nucleic acid, consisting as a whole or in part of peptide motifs possessing a majority of amino acids with a basic character, such as lysine, histidine, arginine (histones, nucleolin, protamine or derivatives thereof) and optionally (iv) a targeting element which makes it possible to orient the transfer of the nucleic acid, such as a ligand of the intracellular type such as a nuclear localization signal sequence (NLS) which favours the accumulation of the transfected DNA inside the nucleus and which may be combined with the peptide compound to form a chimeric peptide comprising a protein fragment (histone or protamine or nucleolin) and an NLS sequence. However, this system requires the presence of a cationic polymer and/or a lipofectant, which have the disadvantage of being toxic and/or costly,
International Application WO 97/30170 which also describes compositions for transfecting eukaryotic cells, which comprise the nucleic acid to be transfected, at least one cationic lipid at a suboptimal concentration and at least one acidic peptide (active on the membrane) which destabilizes the endosomal membrane and thus increases the transfection efficiency. The positive charge/negative charge ratio is between 0 and 3. The selected peptides are derived from the influenza virus, so as to induce effective rupture of the endosomes. The presence of the lipids is necessary in this composition because of the fact that the selected peptide does not allow passage across the first cell membrane.
Such complexes do not therefore make it possible to avoid the disadvantages linked to the use of liposomes.
That is undoubtedly the reason why complexes using only peptides have been provided:
European Patent Application 0,544,292, which describes a complex for transfecting nucleic acid which comprises a fusion protein consisting of a cellular factor (growth factor, viral antigen, toxin, integrin or lipoprotein) and a basic polycationic peptide comprising arginine and/or lysine residues,
International Application WO 94/23751, which describes a transfer peptide which comprises three parts: (1) a ligand L1 (peptide of 2 to 100 amino acids), capable of binding to a binding site at the surface of eukaryotic cells (membrane receptor) (example: peptide RGD, domain for binding of growth factors, hormones, viral antigens or lipoproteins), (2) a ligand L2 similar to L1 (peptide of 2 to 20 amino acids), which binds to the outer nuclear membrane of eukaryotic cells, such as an NLS sequence and (3) a ligand L3 corresponding to a basic peptide (3 to 100 amino acids) (histone fragment H1 or H2B, for example). The transfer peptides described in this Application therefore have a general ligand structure for a membrane receptor-ligand for the outer nuclear membrane-basic peptide. Such a structure was proposed in order to improve the specificity of the complex towards target cells, but has a toxicity level of the same order as that of the liposomes; in addition, the construct should be adapted as a function of the target cells (presence of specific receptors on the target cells), and
International Application WO 95/31557 which describes a transfection vector comprising a synthetic peptide and the nucleic acid to be transfected. The synthetic peptide comprises a polymeric chain of basic amino acids, preferably at the C-terminal position (10-50 amino acids, such as lysine, arginine and ornithine), an NLS peptide (6-15 amino acids, such as the NLS sequence of the SV40 T antigen, the NLS sequence of the polyoma T antigen, the NLS sequence of adenovirus E1a or the NLS sequence of adenovirus E1b, preferably at the N-terminal position and a hinge region of neutral amino acids (6-50 amino acids selected from glycine, alanine, leucine and isoleucine), between the polymeric chain and the NLS peptide. The preferred NLS sequence is the sequence of the SV40 virus T antigen (small sequence of basic amino acids: PKKKRKV SEQ ID NO: 25), which is efficient in mammalian cells or a short hydrophobic sequence which contains one or more basic amino acids (KIPIK SEQ ID NO:43). The hinge sequence comprises 6-26 neutral amino acids selected solely from Gly (G), Ala (A), Leu (L) and Ile (I). The peptide:DNA ratio (by weight) is between 1:1 and 1:10. The peptide described in this document passes across the cell membrane with difficulty and that is the reason why it is recommended, in this International Application, to treat the cells before the transfection: the cells are treated with a hypertonic solution, and then with a hypotonic solution in the presence of the nucleic acid-peptide complex. The hypertonic solution may contain PEG (0.3 M-0.6 M) and sucrose (10-25%).
These various complexes possess the property of condensing the DNA and of promoting its combination with the cell membrane; however, they are far from being as efficient as the viral vectors, in particular because of an insufficient condensation of the DNA to be transfected and/or of the difficulties encountered by the transfected DNA in coming out of the endosome without being degraded and in penetrating into the cell nucleus.
Seeking to develop novel vectors not exhibiting the disadvantages of the viral vectors, the inventors developed the vector described in International Application WO 97/18317, which describes compositions comprising an adenoviral protein complex consisting:
A. of an adenoviral protein complex, namely:
either of 12 pentons, each comprising at least one fibre and one penton base, excluding any other constituent element of the genome of an adenovirus, which fibre(s) and penton base are derived either from the same adenovirus, or from different adenoviruses, the said pentons being linked by the penton bases and forming a dodecahedron structure, stable to proteolytic enzymes, which complex has a molecular weight of between 4.8xc3x97106 and 6.6xc3x97106;
or of 12 penton bases, excluding any other constituent element of the genome of an adenovirus, which penton bases are derived either from the same adenovirus, or from different adenoviruses, and form a dodecahedron structure, stable to proteolytic enzymes and in that it has a molecular weight of between 3.2xc3x97106 and 4xc3x97106.
B. of a nucleic acid sequence to be transfected, and
C. of a ligand between the adenoviral protein complex and the nucleic acid, such as peptides whose N-terminal portion comprises the N-terminal amino acid sequence of a fibre of an adenovirus of any serotype (region for attachment to the adenoviral protein complex) and whose C-terminal portion comprises a polylysine or a polyarginine.
The transfection vectors described in this Application allow the internalization of the nucleic sequence to be transfected and increase the permeability of the endosomes; this is, however, a relatively complex structure which mimics the behaviour of adenoviruses; indeed, the adenoviral particles are relatively complex and comprise several substructures; in particular the outer part or capsid is formed predominantly of three proteins: the hexon, the penton base and the fibre; the fibre allows the attachment of the virion to a cell receptor, whereas the penton base allows the internalization of the virion.
Continuing their research studies, the inventors have found that, unexpectedly, a peptide derived from the adenovirus fibre protein is capable of efficiently transfecting nucleic acid sequences or proteins, in the absence of liposomes and of the treatment of cells.
The subject of the present invention is a peptide vector for transfecting a chemical substance selected from the group consisting of nucleic acid sequences, proteins, peptides and pharmacologically active chemical substances, characterized in that it contains, in addition to the said chemical substance, at least one transfecting peptide derived from the whole or part of an adenovirus fibre and comprising at least one region consisting of at least 50% of hydrophobic amino acids selected from the group consisting of alanine, valine, phenylalanine, isoleucine, leucine, proline and methionine.
In accordance with the invention, the said peptide is derived from a fibre of an adenovirus selected from the group consisting of Ad2, Ad3, Ad4, Ad7, Ad8, Ad9, Ad11, Ad12, Ad15, Ad16, Ad21, Ad40, Ad41, FAV1 (CELO) and FAV7 or from a fragment of the SV40 virus Vp3 protein.
According to an advantageous embodiment of the said transfection vector, the said transfecting peptide comprises at least:
a segment of an NLS sequence derived from an adenovirus fibre comprising between 4 and 5 amino acids and including a sequence selected from the group consisting of the following sequences: X0-Lys-Arg-Val-Arg (X0KRVR) (SEQ ID NO:1), X0-Lys-Arg-Ala-Arg (X0KRAR) (SEQ ID NO:2), X0-Lys-Arg-Ser-Arg (X0KRSR) (SEQ ID NO:3), X0-Lys-Arg-Leu-Arg (X0KRLR) (SEQ ID NO:4), X0-Lys-Arg-Thr-Arg (X0KRTR) (SEQ ID NO:5), X0-Pro-Lys-Lys-Pro-Arg (X0PKKPR) (SEQ ID NO:6), in which X0 is zero or represents Thr (T), Ala (A), Ser-Lys (SK) or Ser (S), or a segment of the SV40 virus Vp3 protein and in particular the sequence GPNKKKRKL (SEQ ID NO:24),
a hydrophobic sequence comprising between 7 and 50 amino acids, derived from an adenovirus fibre and selected from the group consisting of at least one of the following sequences X1-Phe-Asn-Pro-Val-Tyr-Pro-Tyr-X2 (X1FNPVYPYX2) (SEQ ID NO:7), X1-Phe-Asp-Pro-Val-Tyr-Pro-Tyr-X2 (X1FDPVYPYX2) (SEQ ID NO:8), in which:
X1 is zero or represents a sequence of at most 43 amino acids, preferably a sequence of 5 to 15 amino acids, comprising hydrophobic and/or polar and/or acidic charged amino acids, and in particular one of the following sequences: Leu-Ser-Asp-Ser (LSDS) (SEQ ID NO:9), Leu-Ser-Thr-Ser (LSTS) (SEQ ID NO:10), Leu-Ser-Ser-Ser (LSSS) (SEQ ID NO:11), Pro-Ser-Glu-Asp-Thr (PSEDT) (SEQ ID NO:12), Val-Asp-Asp-Gly (VDDG) (SEQ ID NO:13), Thr-Gln-Tyr-Ala-Glu-Glu-Thr-Glu-Glu-Asn-Asp-Asp (TQYAEETEENDD) (SEQ ID NO:14) or X3-Glu-Asp-Asp (X3EDD) (SEQ ID NO:15) in which X3 represents Ala (A), Val (V), Leu (L), Phe (F) or Ile (I) and
X2 is zero or represents a sequence of at most 43 amino acids, preferably a sequence of 5 to 15 amino acids, comprising hydrophobic and/or polar and/or charged amino acids, and in particular one of the following sequences: Glu-Asp-Glu-Ser (EDES) (SEQ ID NO:16), Asp-Thr-Glu-Thr (DTET) (SEQ ID NO:17), Asp-Ala-Asp-Asn (DADN) (SEQ ID NO:18), Asp-Pro-Phe-Asp (DPFD) (SEQ ID NO:19), Gly-Tyr-Ala-Arg (GYAR) (SEQ ID NO:20), Glu-His-Tyr-Asn (EHYN) (SEQ ID NO:21), Asp-Thr-Ser-Ser (DTSS) (SEQ ID NO:22) or Asp-Thr-Phe-Ser (DTFS) (SEQ ID NO:23) and
a polymeric sequence of basic amino acids or a cationic polymeric sequence or a polyalcohol.
There are understood by:
hydrophobic amino acids, the following amino acids: Ala, Val, Leu, Ile, Pro, Phe and Met;
acidic charged amino acids, the following amino acids: Asp and Glu;
basic charged amino acids, the following amino acids: Lys, Arg and ornithine; and
neutral polar amino acids, the following amino acids: Gly, Ser, Thr, Cys, Tyr, Asn, Gln, His and Trp.
Advantageously, the said transfecting peptide is branched and comprises at least two fragments which are derived from an adenovirus fibre; the said fragments each comprise a segment of an NLS sequence and a hydrophobic sequence, as defined above and are linked to each other by a polymeric sequence such as a polymeric sequence of basic amino acids.
When the chemical substance is a nucleic acid sequence, it is selected from genes which encode a polypeptide having a therapeutic activity, antisense sequences and ribozymes.
In the case of a coding sequence, it comprises, in addition, an active promoter for the expression of the polypeptide.
The said promoter is in particular selected from the group consisting of constitutive promoters and inducible promoters.
Surprisingly, such a transfecting peptide vector comprising no lipids (in the form of liposomes, for example) or penton base is capable of efficiently transfecting in particular nucleic acid sequences of any size, up to the nucleus and without poisoning the transfected cell.
In all cases, the exogenous nucleic acid sequence, the protein of interest or any other chemical substance, combined with the said transfection vector penetrates into the cell (internalization).
Surprisingly, the transfecting peptide-cell receptor interaction significantly increases both the internalization of the transfection vector and the permeability of the endosomes, which significantly increases the passage of the exogenous nucleic acid, of the protein of interest or of any other chemical substance from the endosomes to the cytoplasm and to the nucleus, in comparison with the use of a vector including lipids (in the form of liposomes, for example).
Such transfecting peptide vectors prove surprisingly more efficient and less toxic than compositions containing liposomes (cationic lipids or lipofectants).
According to another advantageous embodiment of the said transfection vector, the said transfecting peptide comprises at least:
a segment of an NLS sequence derived from an adenovirus fibre comprising between 4 and 5 amino acids and including a sequence selected from the group consisting of the following sequences: X0-Lys-Arg-Val-Arg (X0KRVR) (SEQ ID NO:1), X0-Lys-Arg-Ala-Arg (X0KRAR) (SEQ ID NO:2), X0-Lys-Arg-Ser-Arg (X0KRSR) (SEQ ID NO:3), X0-Lys-Arg-Leu-Arg (X0KRLR) (SEQ ID NO:4), X0-Lys-Arg-Thr-Arg (X0KRTR) (SEQ ID NO:5), X0-Pro-Lys-Lys-Pro-Arg (X0PKKPR) (SEQ ID NO:6), in which X0 is zero or represents Thr (T), Ala (A), Ser-Lys (SK) or Ser (S), or a segment of the SV40 virus Vp3 protein and in particular the sequence GPNKKKRKL (SEQ ID NO:24),
a hydrophobic sequence comprising between 7 and 50 amino acids, derived from an adenovirus fibre and selected from the group consisting of at least one of the following sequences X1-Phe-Asn-Pro-Val-Tyr-Pro-Tyr-X2 (X1FNPVYPYX2) (SEQ ID NO:7), X1-Phe-Asp-Pro-Val-Tyr-Pro-Tyr-X2 (X1FDPVYPYX2) (SEQ ID NO:8), in which:
X1 is zero or represents a sequence of at most 43 amino acids, preferably a sequence of 5 to 15 amino acids, comprising hydrophobic and/or polar and/or acidic charged amino acids, and in particular one of the following sequences: Leu-Ser-Asp-Ser (LSDS) (SEQ ID NO:9), Leu-Ser-Thr-Ser (LSTS) (SEQ ID NO:10), Leu-Ser-Ser-Ser (LSSS) (SEQ ID NO:11), Pro-Ser-Glu-Asp-Thr (PSEDT) (SEQ ID NO:12), Val-Asp-Asp-Gly (VDDG) (SEQ ID NO:13), Thr-Gln-Tyr-Ala-Glu-Glu-Thr-Glu-Glu-Asn-Asp-Asp (TQYAEETEENDD) (SEQ ID NO:14) or X3-Glu-Asp-Asp (X3EDD) (SEQ ID NO:15) in which X3 represents Ala (A), Val (V), Leu (L), Phe (F) or Ile (I) and
X2 is zero or represents a sequence of at most 43 amino acids, preferably a sequence of 5 to 15 amino acids, comprising hydrophobic and/or polar and/or charged amino acids, and in particular one of the following sequences: Glu-Asp-Glu-Ser (EDES) (SEQ ID NO:16), Asp-Thr-Glu-Thr (DTET) (SEQ ID NO:17), Asp-Ala-Asp-Asn (DADN) (SEQ ID NO:18), Asp-Pro-Phe-Asp (DPFD) (SEQ ID NO:19), Gly-Tyr-Ala-Arg (GYAR) (SEQ ID NO:20), Glu-His-Tyr-Asn (EHYN) (SEQ ID NO:21), Asp-Thr-Ser-Ser (DTSS) (SEQ ID NO:22) or Asp-Thr-Phe-Ser (DTFS) (SEQ ID NO:23), which transfecting peptide is combined with a polymeric sequence of basic amino acids, a cationic polymer or a polyalcohol.
According to another advantageous embodiment of the said transfecting peptide vector, the polymeric sequence of basic amino acids comprises between 10 and 50 amino acid residues, selected from the group consisting of lysine, arginine and ornithine.
According to another advantageous embodiment of the said transfecting peptide vector, the cationic polymeric sequence is selected from the group consisting of polyamines and quaternary ammonium polymers; a preferred polyamine is polyethyleneimine (PEI).
In accordance with the invention, the said polyalcohol is preferably a C3-C20, and in particular glycerol or dextrans.
According to another advantageous embodiment of the said transfecting peptide vector, the NLS sequence is at the N-terminal end of the transfecting peptide and the polymeric sequence of basic amino acids is at the C-terminal end of the said transfecting peptide.
According to another advantageous embodiment of the said transfecting peptide vector, when the chemical substance is a nucleic acid, the transfecting peptide/nucleic acid ratio is between 0.3:1 and 15:1, preferably between 2:1 and 6:1, preferably between 4:1 and 6:1.
According to another advantageous embodiment of the said transfecting peptide vector, it is combined with a targeting ligand.
The subject of the invention is also a composition, characterized in that it essentially consists of a transfer vector as defined above and a suitable vehicle selected from the group consisting of bile salts, antiproteases, cyclodextrins and derivatives thereof, antiseptics and polyols.
The compositions according to the invention have many applications as medicaments, in human and veterinary medicine:
in human and-animal gene therapy, in particular in hereditary diseases,
as antiviral agents (antisense sequences or ribozymes),
as immunogenic or vaccinal agents,
as antibacterial or anticancer agents, and the like.
The subject of the present invention is also a method of transfecting eukaryotic cells in vitro with a chemical substance selected from the group consisting of nucleic acid sequences, proteins, peptides and pharmacologically active chemical substances, characterized in that it comprises the bringing into contact and the incubation of a transfecting peptide vector in accordance with the invention, in a dilution buffer comprising 100-150 mM NaCl with eukaryotic cells for 15 to 120 minutes at room temperature, the chemical substance to be transfected:transfecting peptide ratio being between 0.3:1 and 15:1, preferably between 2:1 and 6:1, preferably between 4:1 and 6:1.